The CCAAT Binding Factor CBF, also known as NFY, is a ubiquitous transcription factor which binds to the proximal promoters of the two type I collagen genes and to cognate sites in many other eucaryotic promoters, including promoters that are regulated during the cell cycle. CBF is composed of three different subunits CBF-A, CBF-B and CBF-C; all three are needed for DNA binding. CBF-A and CBF-C contain a histone-fold motif and whereby these two subunits stably interact with each other; this heterodimer interacts with the third subunit CBF-B to form the CBF heterotrimer, which binds with high affinity to CCAAT motifs in promoters. CBF-B and CBF-C contain transcriptional activation domains which act additively. This application is based on the hypothesis that CBF is a critical transcription factor for a number of genes including genes that are controlled during the cell cycle and the products of which are themselves involved in the cell cycle. In a first part, this application proposes genetic approaches to examine the function of CBF in vivo in cartilages and cartilage primordia of intact mice and embryos. This will be achieved (a) by creating transgenic mice in which a dominant-negative CBF mutant is expressed in these tissues under the control of chondrocyte-specific Col2alpha1 regulatory elements and (b) by generating mice in which the endogenous CBF-B gene is conditionally inactivated in cartilages. We speculate that in mutant mice the growth of these tissues will be affected. Our experiments will create a series of endogenous and transgenic mutant CBF-B alleles in mouse embryos. By using methods that measure levels of many RNAs simultaneously in specific tissues of wild type and mutant mice and/or embryos, the identify and extent of genes controlled by CBF will be examined. The second part of this application proposes biochemical approaches to analyze the function of CBF in vitro using a reconstituted chromatin template that resembles the natural cellular substrate for transcription. It is based on the hypothesis that CBF plays a key role in producing changes in chromatin structure in genes that are controlled by this transcription factor. In the mouse Col1alpha2 promoter, which will be used as a model system, changes in chromatin structure occur around the CBF binding site in cells which express this gene. These experiments will examine whether in the reconstituted chromatin system CBF, eventually in cooperation with other proteins, produces changes in chromatin structure that are essential for promoter activation.